USING ULTRAFILTRATION TO FACILITATE SIMULTANEOUS QUANTIFICATION OF 5-FLUOROURACIL IN MOUSE PLASMA AND TISSUES BY HPLC

Abstract

A simple and rapid HPLC method that includes ultrafiltration to remove plasma and tissue interference component was developed to determine 5-Fluorouracil (5-FU) concentration in mouse plasma and tissues (heart, liver, spleen, lung, and kidney). Following ultrafiltration, standards, quality controls, and plasma samples were separated by reversed-phase HPLC on an ODS analytical column and quantified using UV detection at 265 nm. The mobile phase was 5 mM phosphate buffer (pH 5)-methanol (95:5, v/v), with a flow-rate of 0.6 mL/min. The calibration curves for 5-FU in mouse plasma and tissue samples were all linear (r² > 0.997) over the drug concentration range of 40 to 25000 ng/mL. For all plasma and tissue samples, both the intra- and inter-day precisions were less than 5% expressed in RSD, and the accuracy was between 96 and 104%. The limit of detection was 20 ng/mL. The overall recoveries were >73%. The method was illustrated with a preliminary in vivo analysis on 3 mice treated with intravenous injection of 5-FU, and most of the drug was found deposited in mouse liver 1 hour after the administration.

Keywords:

• biodistribution
• deproteinization
• endogenous interference
• in vivo analysis
• ODS column
• UV detection

ACKNOWLEDGMENTS

This work was supported by both Shandong Provincial Natural Science Foundation (grant number ZR2010HL067) and JMC Science and Research Project Startup Fund for PhD Faculty.

Notes

^a y = Ax + B with a weighting of 1/y; y is the peak area ratio; x, the concentration of 5-FU; A, the slope; B, the intercept. The value of each parameter is the mean value, and the value in each pair of parentheses is RSD (%).

^a The value of each parameter is the mean value, and the value in each pair of parentheses is RSD (%).

^a The stability test results of 5-FU in the plasma and tissue ultrafiltrates at room temperature and at −20°C was assessed by spiking mouse plasma and tissues with 5-FU at 40, 1000, and 25000 ng/mL.

^b The value of each parameter is the mean value, and the value in each pair of parentheses is RSD (%).